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Abstract

We assessed the agreement between sampling windows of different size and orientation on packing density estimates in images of the parafoveal cone mosaic acquired using a flood-illumination adaptive optics retinal camera. Horizontal and vertical oriented sampling windows of different size (320x160 µm, 160x80 µm and 80x40 µm) were selected in two retinal locations along the horizontal meridian in one eye of ten subjects. At each location, cone density tended to decline with decreasing sampling area. Although the differences in cone density estimates were not statistically significant, Bland-Altman plots showed that the agreement between cone density estimated within the different sampling window conditions was moderate. The percentage of the preferred packing arrangements of cones by Voronoi tiles was slightly affected by window size and orientation. The results illustrated the high importance of specifying the size and orientation of the sampling window used to derive cone metric estimates to facilitate comparison of different studies.

Figures (6)

Performance of the cone identification algorithm. Shown are images from three cases acquired at the nasal location (upper row). Scale bar is 25 µm. Red crosses represent cones identified by the algorithm, blue and yellow squares indicate those added and removed by the user respectively (lower row). The average number of cones added manually across all images within the vertical oriented sampling windows of 160x80 µm size was 0.8 ± 0.6%. In general, misidentified cones were more frequently located near the edge of the sampling windows (boundary effect). Cones whose edges were, also in part, outside the image section were not labelled.

Bland-Altman plots showing the agreement between cone density values calculated within horizontal and vertical oriented sampling windows of same size at the 1.20 degree nasal retinal location. Average and difference density values between sampling windows are plotted in the x- and y-axes respectively. The presentation of the 95% limits of agreement is for visual judgement of how well two methods of measurement agree. The smaller the range between these two limits the better the agreement is. The average difference and the distribution of points across the diagram, however, provide further information on the agreement between the two measurements. A wide 95% CI has been calculated between sampling windows of different orientation and same size.

Bland-Altman plots showing the agreement between cone density values calculated within vertical (left column) and horizontal (right column) oriented sampling windows of different sizes. Average and difference density values between sampling windows are plotted in the x- and y-axes respectively. Cone density values estimated within the vertical oriented windows of 320x160 µm and 160x80 µm showed low average difference and small 95% CI. The average differences calculated between windows of different sizes increased as the window size decreased. The 95% CI was wider between the horizontal oriented windows of different size than the vertical ones, except for the horizontal windows of 80x160 µm and 40x80 µm.

Voronoi maps obtained from cone coordinates estimated within the three sampling vertical window conditions at the nasal fixation location in two subjects (ML_05 and ML_06). The percentage of 6n arrangements (green tiles) is 50.9% and 47.5% within the 160x320 µm windows respectively. It was 56.2% and 46.4% within the 80x160 µm windows and 50.6% and 51.3% within the 40x80 µm windows respectively. In subject ML_06 (i.e., the case showing the lower % of 6n arrangement), the percentage of 5n arrangement (yellow tiles) increased from 26.6% to 31.9% from the largest to the smallest sampling window. The corresponding images of the cone mosaic at the same retinal location are shown for subject ML_06. The boundary effect may influence the estimation of the preferred packing arrangement of cones near the edge of the image section. The algorithm’s performance and the subsequent manual check to identify cones are additional sources of error for accurate reconstruction of a Voronoi map.

Voronoi maps obtained from cone coordinates estimated within the three sampling horizontal window conditions at the temporal fixation location in two subjects (ML_08 and ML_07). Across the horizontal oriented sampling window, the percentage of 6n arrangement tended to increase with decreasing area. In these cases, it ranged from 49.7% and 49.8% to 53.3% and 46.6% respectively. In subject ML_07, the percentage of each of the preferred cone packing arrangements showed differences ≤2.3% between the three sampling window conditions. The images of the cone mosaic at the same retinal location are shown for subject ML_07.